Increased PCR sensitivity by using paraffin wax as a reaction mix overlay

Abstract

The use of mineral oil as a reaction mix overlay in conventional PCR may lead to problems. In addition to more difficult handling, traces of mineral oil in amplicon suspensions have been shown to decrease the efficiency of post-PCR manipulations. Commercial alternatives aimed at resolving the problem more than double the cost of an amplification. This is an important drawback when dealing with a large number of clinical samples. The use of inexpensive paraffin wax as a reaction mix overlay eliminated problems associated with the presence of mineral oil while being more practical and safer in handling potentially contaminated clinical samples. Moreover, when used in conjunction with a modified hot start technique, the use of paraffin wax increased the specificity and sensitivity of PCR amplifications over mineral oil in similar hot start conditions. Using the aforementioned method, the increase in specificity and sensitivity has enabled specific detection of viral DNA in clinical samples which the conventional PCR method failed to detect.